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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source: Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Reasons for the selection of the study system: Activated sludge from the sewage plant at Hildesheim is well suited as it comprises mostly municipal sewage and hardly industrial chemical waste.
- Colony forming units in the test vessels: 10e4- 10e5CFU/L
- Pretreatment: filtering with folded filter: first 200 mL filtrate are not used, the second filtrate is used to initiate inoculation.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Initial conc.:
9.44 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral nutrient solution according to OECD 301 D
- Test temperature: 20°C
- pH mean: 7.44
- Frequency and duration of the application: Once per setting up over 28 d

TEST SYSTEM
- Test vessels: BOD bottles with glass stoppers (volume 300 mL)
- 10 bottles containing test item and inoculum (test suspension)
- 10 bottles containing only inoculum (inoculum control)
- 10 bottles containing reference compound and inoculum (functional control)
- 10 bottles containing test item and reference compound and inoculum (toxicity control)

CONTROL AND BLANK SYSTEM
- Inoculum control: Nutrient solution and inoculum
- Toxicity control: 2 mg/L test item + 5.0 mg/L functional control item + nutrient solution and inoculum

VALIDITY:
The percentage degradation of the functional control must reach the pass level of 60 % by day 14.
The oxygen depletion in the inoculum control should not exceed 1.5 mgO2/L after 28 d.
The residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L at any time.
The difference of extremes of replicate values of removal of the test item at the end of the test, at the plateau or at the end of the 10-d-window as appropriate must be less than 20 %, if the test item is readily biodegradable.
If in the toxicity test containing both test item and reference item, less than 25 % degradation (based an COD) occurred within 14 days, the test can be considered to be inhibitory.

EVALUATION:
For the calculation of the BOD and the biodegradation of the test item, functional control and toxicity control the oxygen depletion of the control was taken into account in the calculations. The calculations were performed according to the OECD-guideline 301 D.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
77
Sampling time:
14 d
Remarks on result:
other: test item
Details on results:
The ThOD of 2.36 mgO2/mg test item was used for calculation of BOD and biodegradation in %.
The test item reached the pass level of a biodegradation rate > 60 % very rapid after 6 days. The residual concentration of oxygen in the test bottles was a mean of 0.65 mg 02/L after 28 days. The difference of extreme of replicate values was less than 20 % at the end of testing.
Results with reference substance:
Results of the inoculum control: The oxygen demand in the inoculum control exceeded the recommended 1.5 mg02/L and came to a maximum of 1.68 mg02/L after 28 days. The insignificantly higher demand is considered to have no effect on results.

Results of the functional control: The pass level of a biodegradation > 60 % was achieved after 6 days. The validity criterion that the degradation should be > 60 % alter 14 d was fulfilled. The residual concentration of oxygen fall negligible below 0.5 mgO2/L in one replicate after 21 and 28 days.

Results of the toxicity control: In the toxicity control the recommended rate of a biodegradation of 25 % was reached after 4 days. The biodegradation achieved a maximum rate of 85% aller 14 days. The biodegradation of the reference item was not inhibited by the test item. Due to the rapid biodegradation the residual oxygen in the test bottles fell below 0.5 mg/L after 14, 21 and 28 days. lt is considered that this effect has no impact on the results.

Biodegradation of the test item in comparison to the functional control and the toxicity control:

  biodegradation (%) study day (d)
 7 14  21  28 
  test item 4 mg/L   
75  77  68  70 
 functional control 10 mg/L  
 74 85  87  85 
 toxicity control 2 + 5 mg/L  
 77 85  82  83 

The test item reached the 10 % level after 1 day and the biodegradation was very rapid. The pass level of biodegradation > 60 % was reached in the 10-d-window. The biodegradation achieved a maximum of 77 % after 14 days. At this point of time the plateau was reached.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item must be regarded to be readily biodegradable as it reached >60% biodegradation after 6 days and a maximum of 77% degradation after 14 days and the 10d-window was met.
Executive summary:

In the current test the ready biodegradability of the test item was assessed with the Closed Bottle Test with a non adapted activated sludge over a period of 28 days.

The degradation was followed by measuring the oxygen concentrations. The oxygen concentration was depleted by the respiration of bacteria. The biodegradation is therefore expressed as the percentage BOD and was calculated for each oxygen analysis.

The oxygen depletion in the inoculum control reached a maximum of 1.68 mgO2/L until day 28.

In order to check the activity of the test system sodium acetate was used as functional control. The functional control reached the pass level > 60 % after 6 days. After 14 days a biodegradation rate of 85 % and the plateau was reached.

In the toxicity control containing both test and reference item a maximum of 85 % degradation occurred within 14 days. The degradation of the reference item seemed not to be inhibited by the test item. The test item was not inhibitory at tested concentration of 2 mg/L.

The test item reached the 10 % level after 1 day. The biodegradation of the test item was very rapid and came to a maximum of 77 % after 14 days. At this point of time the plateau was reached.

The test item must be regarded to be readily biodegrable according to CLP criteria.

Description of key information

The test item must be regarded to be readily biodegrable according to CLP criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One study is available assessing the ready biodegradability of the test item via the Closed Bottle Test.

The degradation was followed by measuring the oxygen concentrations. The oxygen concentration was depleted by the respiration of bacteria. The biodegradation is therefore expressed as the percentage BOD and was calculated for each oxygen analysis.

The biodegradation of the test item was rapid and the 10-d-window was reached. The biodegradation came to a maximum of 77 % after 14 days. At this point of time the plateau was reached.

Comparing the results to the criteria set out in the CLP regulation EC No 1272/2008 in Annex 1 section 4.1.2.9 the test item is considered to be ready biodegradable as it meets the criteria.